The present invention relates to DNA plasmids to be used for the production of recombinant proteins. More specifically, the present invention concerns the addition of specific DNA elements to expression plasmids that serve a function as enhancing elements. The outcome is to improve the yields of recombinant protein production.
There are a number of different strategies for the large-scale production of recombinant proteins to be used in, for example, the pharmaceutical industry. In certain cases it is desirable that the recombinant protein is made in eucaryotic hosts. These hosts may be cultivated cells or animals made transgenic with respect to the gene of interest. In the latter situation, transgenic expression in milk is a valuable technique since transgenes, active in the mammary gland, have been described and milk is a readily available body fluid.
The present invention relates to, in an unrestricted way, an improvement in expression vectors used to produce recombinant proteins in milk. These improved expression vectors will increase the yield of valuable recombinant proteins which will be of value for the facilitation of subsequent handling and purification steps.
Construction of a transgene requires certain basic ingredients, one being the structural gene containing the coding information for the protein of interest. A basal eucaryotic gene expression promoter is also required. In addition, other sequences can be used that confer tissue specificity or enhance expression in response to stimulus. The present invention relates to a specific type of enhancers, namely enhancers responding to hormonal stimuli. The particular enhancer in question is a sequence of DNA that confers a response to signals evoked by pituitary hormones belonging to the group of lactogenic hormones such as prolactin (Prl) and placenta lactogen (PL) and somatogenic hormones such as growth hormone (GH). Both of these groups of hormones occupy central roles in the stimulation of mammary gland development and function. The present invention concerns the definition of enhancers responding to both lactogenic and somatogenic hormones and the construction of expression vectors, that, in their ability to respond to both lactogenic and somatogenic hormones, will function in an improved manner as transgenes for production of recombinant proteins in milk.
Previous studies have defined a gene, the Serine Protease Inhibitor 2.1 (SPI) gene, that responds to GH. In the 5xe2x80x2 flank of this gene a DNA element has been identified that enhances gene expression in a GH-dependent fashion. The sequence of this GH response element (SPI GH-RE) in question is: SEQ ID NO:1 GATCTACGCTTCTACTAATCCATGTTCTGAGAAATCATC CAGTCTGCCCATG, (Yoon et al. J. Biol. Chem. 265; 19947 (1991)) Within this sequence we now disclose a shorter xe2x80x9cSPI-GAS like elementxe2x80x9d; TTCTGAGAA, that constitutes the core GH regulated sequence. As exemplified below the SPI-GAS element is also functional when transferred to a reporter gene such as the Luciferase gene (Sliva D. et al J. Biol.. Chem. in press). In the following we also disclose that the GH-regulated sequences described above are also regulated by prolactin and that this can be used to design new expression vectors that improve existing vectors used to produce recombinant proteins in milk.